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An improved silver impregnation method for tracing degenerating nerve fibers and their terminals in frozen sections
Brain Research, 82 (1974) 279-283
279
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
An improved silver impregnation method for tracing degenerating nerve fibers and their terminals in frozen sections
C. KALAHA-BRUNST, R. A. GIOLLI* ANDD. J. CREEL Neuropsychology Research Laboratory, Veterans Administration Hospital, Phoenix, Ariz. 85012 and *Departments of Anatomy andPaychobiology, California College of Medicine, University of California, Irvine, Calif. 92664 (U.S.A.)
(Accepted September 17th, 1974)
Silver impregnation of degenerating nerve fibers in frozen sections has usually been initiated by treatment of the sections with an oxidizing agent such as potassium permanganate2,4, 5, phosphomolybdic acid z, or phosphotungstic acid 4. We have tried these oxidants and their associated methods together with several modifications of these methods 1-3,6. None of these earlier procedures yielded impregnations as selective, intensive, consistent, or in other ways satisfactory for the purpose of tracing degenerating fibers as were those obtained by the method to be described below. The method we now use employs an acidified, weakly alcoholic, solution of Mayer's hemalum (hematein is the oxidant) as the pretreatment step. It has consistently produced a superior selective impregnation of the degenerating nerve fibers and axon terminals of several different fiber pathways in the brains of rats, rabbits and squirrel monkeys. Examples of the results which have been obtained with it are illustrated in Figs. 1a-e. Fig. 1a shows that this method allows for a clear visualization, even at a low magnification, of the fiber degeneration present in the primary optic system of the rat following unilateral eye enucleation. Figs. 1b and c are details from Fig. la and depict, respectively, degenerating fibers of passage within the optic pathway, and a zone of degenerating boutons within the dorsal lateral geniculate nucleus. Fig. ld illustrates some degenerating fibers of passage traversing the anterior pretectal nucleus of the rabbit following a lesion to the superior colliculus, and Fig. le shows a zone of degenerating boutons within the most caudal portion of the pulvinar from the same histological slide. The present method has been used with success in demonstrating degenerating cortico-cortical fibers and their terminals within the visual cortex of the squirrel monkey, degenerating cortico-fugal and tecto-thalamic fibers in the rabbit, and degenerating primary optic fibers in the rat. In all of these cases, the method has provided a more thorough silver impregnation of the degenerating fibers and their terminals than has any of the other methods tried 1-3,6. The microscopic examination of the sections has been facilitated by the sharp contrast which exists between the impreg-
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nated structures and the light-colored background in which staining of glial cells and impregnation of normal nerve fibers are suppressed. This method is worthy of being added to the already extensive armamentarium because of its simplicity, consistency and superior results. (1) Fix the brain material by perfusion with a physiological saline solution followed by a 10 ~ buffered neutral formalin solution. Remove the brain the following day and store it in 10 ~ buffered neutral formalin. (2) Several days later, transfer the material to a 25 ~ solution of sucrose in 10 buffered neutral formalin and leave until the tissue sinks to the bottom of the container. (3) Cut the brain on a freezing microtome at a thickness of 20-40 # m and assemble the sections in 2 ~ buffered neutral formalin. (4) When ready to process, sections are rinsed in distilled water and flattened. (5) Stain sections in an acidified solution of Mayer's hemalum for 5 rain. They will become light purple in color. Mayer's hemalum:
1 g hematein (Harleco 232, Harleco Chem., Philadelphia) 19 ml absolute ethanol 50 g aluminum potassium sulfate 1000 ml distilled water 2 ml glacial acetic acid (6) Wash sections in two changes of distilled water. (7) Transfer sections to a 20 ~ aqueous uranyl nitrate solution for 10 min. (8) Wash sections, now light purple to taupe in color, in two changes of distilled water. (9) Transfer sections to a 20 ~ aqueous silver nitrate solution for 10 min. (10) Wash sections, now taupe in color, in two changes of distilled water. (11) Transfer sections to a fresh ammoniacal silver nitrate solution for 3 min. During this step the regions of gray matter will turn bluish-gray while the fiber pathways appear yellow. Ammoniacal silver: 80 ml 5 ~ aqueous silver nitrate 40 ml absolute ethanol 20 ml 58 ~ ammonium hydroxide 6 ml 2.5 ~ aqueous sodium hydroxide In a 250 ml flask mix the silver nitrate solution and the ethanol; cover and set aside.
Fig. 1. a: fiber degeneration within the primary optic fiber system contralateral to ocular enucleation in a rat. The location of Figs. lb and c, both showing details of this photomicrograph, is indicated by arrows, x 11. b: detail from la showing degenerating fibers of passage within the superior quadrigeminal brachium. This photomicrograph is reversed relative to la. x 225. c: detail from la which depicts a zone of boutonal degeneration within the dorsal lateral geniculate nucleus (LGd). x 225. d: a group of degenerating fibers of passage is shown traversing the anterior pretectal nucleus of a rabbit following a lesion to the ipsilateral superior colliculus. × 188. e: a zone of boutonal degeneration within the most caudal portion of the ipsilateral pulvinar of the rabbit. The photograph is taken from the same section as illustrated in Fig. ld. x 188.
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In a 100 ml graduated cylinder pour the ammonium hydroxide, and to it add the sodium hydroxide. Then pour the contents of the cylinder into the flask and swirl until the brown precipitate has fully disappeared. Pour the liquid into a staining dish, add the sections and cover for 3 rain. The solution is made up fresh for each group of sections. (12) Transfer the sections to two dishes of reducer solution for 1 min each and agitate; sections will turn a brownish-yellow color. Reducer solution: 400 ml distilled water 45 ml absolute ethanol 13.5 ml aqueous 1 ~ citric acid 13.5 ml 10 ~ non-neutralized formalin (10 ml 37 ~ formaldehyde and 90 ml distilled water) (13) Wash sections in distilled water. (14) Treat sections in 1 Y/ooaqueous sodium thiosulfate for 1 min. (15) Wash sections in two changes of distilled water for 1 min each. (16) Tone sections in 1% acid alcohol (1 ml 37 ~ hydrochloric acid and 100 ml 70 ~ ethanol) for 0.5 min. During this step the background of the sections becomes honey-colored. (17) Wash sections in three changes of distilled water, mount on albuminized slides, blot with bibulous paper and expose to air for 5 min. Then dehydrate, clear and cover with a synthetic mounting medium. To standardize the method, we offer the following suggestions: (i) during the staining procedure the laboratory temperature and solution temperatures should be kept below 70 °F (21 °C); (ii) the sections can be processed through the sequence in numbers by the use of staining nets; (iii) regardless of the manner used to transfer the sections from one solution to the next, it is important that they be kept flat throughout the procedure to facilitate even staining and to ensure that the sections remain intact; (iv) when staining nets are used, the rinsing fluids should be changed for each group of sections; (v) the Mayer's hemalum will last for several months and need be replaced only when it produces a weak stain; (vi) the solutions used in steps 7 and 9 can be re-used for 10 days while those used in steps 14 and 16 are prepared fresh daily. The authors wish to thank Dr. Johannes Tigges for his consultation with the method and for providing the primate brain material. The technical advice of Mr. Ed Busch of Harleco Chemicals and the assistance of Mr. Hussein Kabil and Mr. David Haste is gratefully appreciated. 1 EAGER, R. P., Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 2 FINK, R. P., AND HEIMER, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 3 GIOLLI, R. A., AND POPE, J. E., The mode of innervation of the dorsal lateral geniculate nucleus
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and the pulvinar of the rabbit by axons arising from the visual cortex, J. comp. Neurol., 147 (1973) 129-144. 4 JOHNSTONE, G., AND BOWSHER, D., A new method for the selective impregnation of degenerating axon terminals, Brain Research, 12 (1969) 47-53. 5 NAUTA, W. J. H., AND GY6AX, P. A., Selective impregnation of degenerating axon terminals in the central nervous system: (1) technic, (2) chemical notes, Stain Technol., 26 (1951) 5-11. 6 NAUTA, W. J. H., AND GY~AX, P. A., Silver impregnation of degenerating axons in the central nervous system: a modified technic, Stain Technol., 29 (1954) 91-93.
279
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
An improved silver impregnation method for tracing degenerating nerve fibers and their terminals in frozen sections
C. KALAHA-BRUNST, R. A. GIOLLI* ANDD. J. CREEL Neuropsychology Research Laboratory, Veterans Administration Hospital, Phoenix, Ariz. 85012 and *Departments of Anatomy andPaychobiology, California College of Medicine, University of California, Irvine, Calif. 92664 (U.S.A.)
(Accepted September 17th, 1974)
Silver impregnation of degenerating nerve fibers in frozen sections has usually been initiated by treatment of the sections with an oxidizing agent such as potassium permanganate2,4, 5, phosphomolybdic acid z, or phosphotungstic acid 4. We have tried these oxidants and their associated methods together with several modifications of these methods 1-3,6. None of these earlier procedures yielded impregnations as selective, intensive, consistent, or in other ways satisfactory for the purpose of tracing degenerating fibers as were those obtained by the method to be described below. The method we now use employs an acidified, weakly alcoholic, solution of Mayer's hemalum (hematein is the oxidant) as the pretreatment step. It has consistently produced a superior selective impregnation of the degenerating nerve fibers and axon terminals of several different fiber pathways in the brains of rats, rabbits and squirrel monkeys. Examples of the results which have been obtained with it are illustrated in Figs. 1a-e. Fig. 1a shows that this method allows for a clear visualization, even at a low magnification, of the fiber degeneration present in the primary optic system of the rat following unilateral eye enucleation. Figs. 1b and c are details from Fig. la and depict, respectively, degenerating fibers of passage within the optic pathway, and a zone of degenerating boutons within the dorsal lateral geniculate nucleus. Fig. ld illustrates some degenerating fibers of passage traversing the anterior pretectal nucleus of the rabbit following a lesion to the superior colliculus, and Fig. le shows a zone of degenerating boutons within the most caudal portion of the pulvinar from the same histological slide. The present method has been used with success in demonstrating degenerating cortico-cortical fibers and their terminals within the visual cortex of the squirrel monkey, degenerating cortico-fugal and tecto-thalamic fibers in the rabbit, and degenerating primary optic fibers in the rat. In all of these cases, the method has provided a more thorough silver impregnation of the degenerating fibers and their terminals than has any of the other methods tried 1-3,6. The microscopic examination of the sections has been facilitated by the sharp contrast which exists between the impreg-
280
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281
nated structures and the light-colored background in which staining of glial cells and impregnation of normal nerve fibers are suppressed. This method is worthy of being added to the already extensive armamentarium because of its simplicity, consistency and superior results. (1) Fix the brain material by perfusion with a physiological saline solution followed by a 10 ~ buffered neutral formalin solution. Remove the brain the following day and store it in 10 ~ buffered neutral formalin. (2) Several days later, transfer the material to a 25 ~ solution of sucrose in 10 buffered neutral formalin and leave until the tissue sinks to the bottom of the container. (3) Cut the brain on a freezing microtome at a thickness of 20-40 # m and assemble the sections in 2 ~ buffered neutral formalin. (4) When ready to process, sections are rinsed in distilled water and flattened. (5) Stain sections in an acidified solution of Mayer's hemalum for 5 rain. They will become light purple in color. Mayer's hemalum:
1 g hematein (Harleco 232, Harleco Chem., Philadelphia) 19 ml absolute ethanol 50 g aluminum potassium sulfate 1000 ml distilled water 2 ml glacial acetic acid (6) Wash sections in two changes of distilled water. (7) Transfer sections to a 20 ~ aqueous uranyl nitrate solution for 10 min. (8) Wash sections, now light purple to taupe in color, in two changes of distilled water. (9) Transfer sections to a 20 ~ aqueous silver nitrate solution for 10 min. (10) Wash sections, now taupe in color, in two changes of distilled water. (11) Transfer sections to a fresh ammoniacal silver nitrate solution for 3 min. During this step the regions of gray matter will turn bluish-gray while the fiber pathways appear yellow. Ammoniacal silver: 80 ml 5 ~ aqueous silver nitrate 40 ml absolute ethanol 20 ml 58 ~ ammonium hydroxide 6 ml 2.5 ~ aqueous sodium hydroxide In a 250 ml flask mix the silver nitrate solution and the ethanol; cover and set aside.
Fig. 1. a: fiber degeneration within the primary optic fiber system contralateral to ocular enucleation in a rat. The location of Figs. lb and c, both showing details of this photomicrograph, is indicated by arrows, x 11. b: detail from la showing degenerating fibers of passage within the superior quadrigeminal brachium. This photomicrograph is reversed relative to la. x 225. c: detail from la which depicts a zone of boutonal degeneration within the dorsal lateral geniculate nucleus (LGd). x 225. d: a group of degenerating fibers of passage is shown traversing the anterior pretectal nucleus of a rabbit following a lesion to the ipsilateral superior colliculus. × 188. e: a zone of boutonal degeneration within the most caudal portion of the ipsilateral pulvinar of the rabbit. The photograph is taken from the same section as illustrated in Fig. ld. x 188.
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In a 100 ml graduated cylinder pour the ammonium hydroxide, and to it add the sodium hydroxide. Then pour the contents of the cylinder into the flask and swirl until the brown precipitate has fully disappeared. Pour the liquid into a staining dish, add the sections and cover for 3 rain. The solution is made up fresh for each group of sections. (12) Transfer the sections to two dishes of reducer solution for 1 min each and agitate; sections will turn a brownish-yellow color. Reducer solution: 400 ml distilled water 45 ml absolute ethanol 13.5 ml aqueous 1 ~ citric acid 13.5 ml 10 ~ non-neutralized formalin (10 ml 37 ~ formaldehyde and 90 ml distilled water) (13) Wash sections in distilled water. (14) Treat sections in 1 Y/ooaqueous sodium thiosulfate for 1 min. (15) Wash sections in two changes of distilled water for 1 min each. (16) Tone sections in 1% acid alcohol (1 ml 37 ~ hydrochloric acid and 100 ml 70 ~ ethanol) for 0.5 min. During this step the background of the sections becomes honey-colored. (17) Wash sections in three changes of distilled water, mount on albuminized slides, blot with bibulous paper and expose to air for 5 min. Then dehydrate, clear and cover with a synthetic mounting medium. To standardize the method, we offer the following suggestions: (i) during the staining procedure the laboratory temperature and solution temperatures should be kept below 70 °F (21 °C); (ii) the sections can be processed through the sequence in numbers by the use of staining nets; (iii) regardless of the manner used to transfer the sections from one solution to the next, it is important that they be kept flat throughout the procedure to facilitate even staining and to ensure that the sections remain intact; (iv) when staining nets are used, the rinsing fluids should be changed for each group of sections; (v) the Mayer's hemalum will last for several months and need be replaced only when it produces a weak stain; (vi) the solutions used in steps 7 and 9 can be re-used for 10 days while those used in steps 14 and 16 are prepared fresh daily. The authors wish to thank Dr. Johannes Tigges for his consultation with the method and for providing the primate brain material. The technical advice of Mr. Ed Busch of Harleco Chemicals and the assistance of Mr. Hussein Kabil and Mr. David Haste is gratefully appreciated. 1 EAGER, R. P., Selective staining of degenerating axons in the central nervous system by a simplified silver method: spinal cord projections to external cuneate and inferior olivary nuclei in the cat, Brain Research, 22 (1970) 137-141. 2 FINK, R. P., AND HEIMER, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 3 GIOLLI, R. A., AND POPE, J. E., The mode of innervation of the dorsal lateral geniculate nucleus
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and the pulvinar of the rabbit by axons arising from the visual cortex, J. comp. Neurol., 147 (1973) 129-144. 4 JOHNSTONE, G., AND BOWSHER, D., A new method for the selective impregnation of degenerating axon terminals, Brain Research, 12 (1969) 47-53. 5 NAUTA, W. J. H., AND GY6AX, P. A., Selective impregnation of degenerating axon terminals in the central nervous system: (1) technic, (2) chemical notes, Stain Technol., 26 (1951) 5-11. 6 NAUTA, W. J. H., AND GY~AX, P. A., Silver impregnation of degenerating axons in the central nervous system: a modified technic, Stain Technol., 29 (1954) 91-93.